With the advent of hybridoma technology and the accompanying availability of monoclonal antibodies, the application of such antibodies has escalated into a variety of areas of the biological sciences. For example, monoclonal antibodies have been used for the study of cell surface antigens, for affinity purification of proteins, for histocompatibility testing, for studying various viruses and for radioimmunoassay. More recently, it has been recognized that monoclonal antibodies may have medical application for drug targeting and immunotherapy (Poynton, C. H., and Reading, C. L. (1984) Exp Biol 44:13-33). With the increased application of the antibodies in the biological and medicinal sciences, there has come a concomitant demand for high levels of antibody production.
To date, efforts have been undertaken to develop culture conditions to maximize cell culture growth and thereby increase resultant product yield. Early work in the development of chemically defined animal cell culture media focused on the formulation of such media to achieve rapid cell proliferation (White, P. R. (1946) Growth 10:231-289, and Waymouth, C. (1974) J. Natl Cancer Inst 53:1443-1448). Such media incorporate specific nutrients, especially amino acids, vitamins, purines, and pyrimidines. Today some of the more widely used basal media for mammalian cell cultures include Hams F-12, Dulbecco's modified Eagle's medium (DME), RPMI 1640, and Iscove's modified DME. All of these above-referenced basal media are also supplemented with several trace metals and salts, including the major cations (potassium, sodium, calcium, magnesium and the like) with concentration values near isotonic levels. The role of inorganic nutrition in cell culture is discussed in a number of references including Shooter, R. A., and Gey, G. O. (1952) Br J Exp Pathol 31:98-103; Waymouth, C. (1974) supra; Birch, J. R., and Pirt, S. J. (1971) J Cell Sci 8:693-700; Ham, R. G., Growth of Cells in Hormonally Defined Media, Cold Spring Harbor Conferences on Cell Proliferation, Vol. 9, Sato, Pardee and Sirbashin, eds., 1982.
Culture media have been developed specifically for low serum and serum-free mammalian cell cultures for production of monoclonal antibodies. One such serum-free medium is disclosed in European Patent Publication 076,647, published Apr. 13, 1983. Other media have been developed by changing levels of supplements such as trace elements, vitamin and hormone additives wherein variations in the traditional basal media are slight. References to such media include, for example, Barnes, D., and Sato, G. (1980) Cell 22:649-655; Cleveland, W. L., et al (1983) J Immunol Meth 56:221-234; Iscove, N., and Melchers, F. (1978) J Ex Med 114:923-933; Kawamoto, T., et al (1983) Analytical Biochemistry 130:445-453; Kovar, J., and Franek, F. (1984) Immunology Letters 7:339-345; Murakami, H., et al (1983) Agric Biol Chem 47(8):1835-1840; Murakami, H., et al (1982) Proc Natl Acad Sci USA 79:1158-1162; Muzik, H., et al (1982) In Vitro 18:515-524; and Wolpe, S. D., "In Vitro Immunization and Growth of Hybridomas in Serum-Free Medium", in J. P. Mather, ed., Mammalian Cell Culture, Plenum Press, New York, 1984.
In addition to providing the right kinds and amounts of nutrients, the culture medium must also provide suitable physicochemical conditions. Parameters that are important for clonal growth of hybridoma cell culture include osmolality, pH buffering, carbon dioxide tension, and partial pressure of oxygen. These all must be adjusted to optimal values for multiplication of each type of cell with, preferably, minimal or no amounts of serum and minimal amounts of protein. Other physical factors such as temperature and illumination must also be controlled carefully.
Efforts to increase antibody yield have focused primarily on means to optimize cell growth and cell density. The optimal conditions for cell growth of mammalian cell culture are generally within narrow ranges for each of the parameters discussed above. For example, typical culture conditions for mammalian hybridoma cell culture use a basal culture medium supplemented with nutritional additives, pH in the range of 6.8 to 7.4 at 35-37C.
As a general point of reference, antibody titers from murine hybridoma cell lines are highly variable from cell line to cell line and range typically from 10 to 350 ug/ml (Lambert, K. J., et al (1987) Dev Indust Microbiol 27:101-106). Human monoclonal antibody expression from human/human or human/mouse fusions are also highly variable from cell line to cell line and range typically from 0.1 to 25 ug/ml (Hubbard, R., Topics in Enzyme and Fermentation Biotechnology, chap. 7, pp. 196-263, Wiseman, A., ed, John Wiley & Sons, New York, 1983). These values are indicative of culture conditions that are optimized for cell growth and cell viability.
Another example from the literature documents that, at least for some cell lines, monoclonal antibody production proceeds even after a culture stops growing (Velez, D., et al., (1986) J Imm Methods 86:45-52; Reuveny, S., et al., (1986) ibid at p. 53-59). Thus, one strategy for increasing monoclonal antibody yield has been to develop culture conditions that allow growth of hybridomas to higher cell densities and to recover the antibodies late in the stationary phase of cell culture. Arathoon, W., and Birch, J. (1986) Science 232:1390-1395 reported that a 1,000 liter hybridoma fermentation produced about 80 grams of monoclonal anti-body during the growth phase and another 170 grams of antibody during an extended stationary/death phase. It was not reported the means, if any, by which the stationary phase of growth was extended.
Another approach from the literature to increasing antibody production is to achieve high cell densities by cell recycle or entrapment methods. Examples of these methods include hollow fiber reactors (Altshuler, G. L., et al (1986) Biotechnol Bioeng XXVIII, 646-658); static maintenance reactors C(Feder, J., et al, EPA 83870128.2, published Nov, 17, 1984); ceramic matrix reactors (Marcipar, A., et al (1983) Annals N.Y. Acad Sci 413:416-420); bead immobilized reactors (Nilsson, K., et al (1983) Nature 302:629-630); perfusion reactors (Feder, J., and Tolbert, W. R. (1985) American Biotechnol Laboratory III:24-36); and others. In some cases, a "resting" cell culture state is reported to be achieved by reducing levels of nutrients in the medium (as by reducing serum or protein supplement levels) with antibody production continuing while growth is slowed.
While a variety of methods to increase antibody yield from hybridoma cell culture are being explored, the primary focus is still on the optimization of cell growth. We have discovered that culture conditions for growth optimization and for optimal product expression may differ and that product expression can be increased under conditions of solute stress, created by the addition of certain solutes, notwithstanding the resulting growth inhibitory effects.
The concept of subjecting animal cells, especially mammalian cell cultures, to an environment of solute stress to produce higher product expression yields, such as increased antibody titers, has not been reported. One means for introducing such an environment to the culture is through salt addition which is easily monitored by measuring the osmolality of the culture medium.
Media osmolality for mammalian cell culture is usually held in the range of 280300 (Jakoby, W. B., and Pastan, I. H., Methods in Enzymology, vol. LVIII, "Cell Culture", Academic Press (1979), pp. 136-137). Of course, the optimal value may depend upon the specific cell type. For example, as reported in Tissue Culture, Methods and Applications, edited by Kruse, Jr., P. F. and Patterson, Jr., M. K., Academic Press (1973) p. 704, human lymphocytes survive best at low (about 230 mOsmol/kg), and granulocytes at higher osmolalities (about 330 mOsmo/kg). Mouse and rabbit eggs develop optimally in vivo at around 270 mOsmol/kg, 250-280 mOsmol/kg being satisfactory, while above 280 mOsmol/kg development is retarded. Iscove reports 280 mOsmol/kg to be optimum for growth of murine lymphocytes and hemopoietic cells, and Iscoves modified DME is adjusted for this growth promoting osmolality (Iscove, N. N. (1984) Method for Serum-Free Culture of Neuronal and Lymphoid Cells, pp. 169-185, Alan R. Liss, ed., New York.
The spread of quality control osmolality values on a number of commercially available tissue culture media is provided in a table beginning at page 706 in the Tissue Culture, Methods and Applications reference, supra. The osmolality values given therein reflect the 280-300 range used for mammalian cell culture.
Another means to introduce an environment of solute stress in the cell culture is through the addition of cellular metabolic products, such as lactic acid and ammonia These products are generally known to be growth inhibitory agents and strategies to reduce the level of these products in the culture medium in order to enhance cell growth have been reported. Imamura, T., et al (1982) Analytical Biochemistry 124:353-358; Leibovitz, A. (1963) Am J Hyg 78:173-180; Reuveny, S., et al. (1986) J Immunological Methods 86:53-59; Thorpe, J. S., et al (1987) "The Effect of Waste Products of Cellular Metabolism on Growth and Protein Synthesis in a Mouse Hybridoma Cell Line", Paper #147 presented at American Chemical Society National Meeting, Aug. 30-Sep. 5, 1987, New Orleans, La.--Symposium on Nutrition and Metabolic Regulation in Animal Cell Culture Scale-Up; and Glacken, M. W., et al (1986) Biotechnology & Bioengineering XXVII:137-1389.
Contrary to the teaching in the art which cautions against major adjustments to culture media osmolality and other physicochemical parameters, we have found that introducing an environment of solute stress during fermentation can favor an increase in specific (per cell) antibody expression and/or increased culture longevity which can result in an increase in antibody titer. It is to such a concept that this invention is directed. Briefly, in a preferred embodiment of the invention, an approach to mammalian cell culture which further optimizes yield of antibody production has been developed in which hybridoma cells are cultured under conditions of controlled solute stress. Optionally, the method incorporates prior art advances including the culture of hybrid mammalian cell lines in serum-flee media or in high density culture to reduce costs and facilitate purification.